BRPI0313559B1 - METHOD FOR PRIMARY CELL CROPPING AND VIRUS AMPLIFICATION UNDER SERUM-FREE CONDITIONS - Google Patents

Abstract

The present invention relates to a method for the cultivation of primary cells. The primary cells are cultivated in a serum free medium comprising a factor selected from the group consisting of growth factors and attachment factors. The method for the cultivation of primary cells may be one step in a method for the amplification of viruses, such as poxviruses. According to this latter method the primary cells are cultivated in a serum free medium comprising a factor selected from the group consisting of growth factors and attachment factors. The cells are then infected with the virus and the infected cells are cultivated in serum free medium until progeny virus is produced.

Description

DESCRIPTION REPORT FOR THE METHOD

FOR GROWING PRIMARY CELLS AND AMPLIFYING VIRUSES UNDER SERUM-FREE CONDITIONS.

The present invention relates to a method for culturing primary cells. Primary cells are grown in a serum-free medium that comprises a factor selected from the group consisting of growth factors and adhesion factors. The method for culturing primary cells can be a step in a method for amplifying viruses, such as poxviruses. According to the latter method, cells

primaries are grown in a serum-free medium that comprises a factor selected from the group consisting of growth factors and adherence factors. The cells are then infected with the virus and the infected cells are cultured in serum-free medium until a viral progeny is produced.

. - 15 Background of the Invention

Most viral vaccines such as attenuated or recombinant viruses are produced from cell culture systems. The cells used for the production of viruses / vaccines can be cell lines, that is, cells that grow continuously in vitro, in the form of a suspension culture of a cell isolated in bioreactors or in the form of

a monolayer of a cell support surface of tissue culture flasks or spinner bottles. Some examples for the cell lines used for virus production are: the MRC-5 human fetal lung cell line used for polio virus production and the WI-38 human fetal lung cell line used for virus production measles, mumps virus and rubella virus (MMR II) (Merk Sharp & Dohme).

Not only cell lines, but also primary animal cells are used for vaccine production. An example of primary cells that are used for viral production are chicken embryonic fibroblasts (CEF cells). These cells are used for the production of measles virus and Japanese encephalitis (Pasteur Me2

rieux), mumps virus (produced by Provaccine), rabies virus (produced by Chiron Berhing GmbH & Co.), yellow fever virus (produced by Aprilvax), influenza virus (produced by Wyeth Labs and Smithkline & Beecham) and the modified Vaccinia Ankara virus (MVA).

CEF cells are used frequently, since many viral vaccines are produced by attenuating the virulent virus that causes the disease, by passing them in series in CEF cells. The attenuated virus no longer causes the disease, but it is still capable of stimulating a potent protective immunity against the virulent form of the virus. a

an example of this type of virus is MVA. This virus has a severely restricted replication in humans and most animals. MVA is being developed as a vaccine vector because it can be used to express antigens derived from a variety of disease-causing agents in humans. Attenuated viruses, such as MVA, are preferably not propagated in human cells since there is a concern that the virus may again become competent for replication in cells of human origin. However, viruses that have recovered their ability to replicate in human cells could be a health risk if administered to humans, in particular 20 if individuals have compromised immunity. For this reason, some

attenuated viruses, such as MVA, are strictly produced from CEF cells, if they are intended for use in humans.

In addition, CEF cells are used for viruses that can grow only in said cells. Examples of such viruses are in particular avian viruses such as avipoxviruses, in particular canary poxviruses, ALVAC, poultry poxviruses and NYVAC.

Cell lines and grown primary cells grown under in vitro culture conditions require a special growth and maintenance medium that can support (I) cell replication in the gar30 phase and (II) cell maintenance since the cells they no longer divide, that is, when the cells are in the stationary phase. The commonly used cell culture media comprise a solution rich in

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salts containing vitamins, amino acids, essential trace elements and sugars. Growth hormones, enzymes and active biological proteins needed to support cell growth and maintenance are usually added as a supplement to the medium in the form of a serum product derived from the blood of animals. Examples for serum products derived from the blood of animals are fetal calf serum, chicken serum, horse serum and porcine serum. These serums are derived from fractionated blood, from which red blood cells and white blood cells have been removed. Primary cells, such as CEF cells, are

even more dependent on animal serum sources than cell lines. Thus, primary cells are generally grown in culture media that comprise 5 to 10% serum, in most cases fetal calf serum (FCS).

Animal sera comprise not only factors that are necessary for cell growth, but also factors that are necessary for cells that grow naturally as adherent cells for adhesion to the cell support surface of the culture vessel. Thus, it is critical for adherent cells that sufficient serum is added to the medium to enable them to grow and form a monolayer.

Unfortunately, bovine / fetal calf serum, as well as other sera from other animals, may contain adventitious pathogens such as viruses. There is a potential risk that these pathogens will be transmitted to the animal / human being that will be treated or vaccinated with the vaccine or other pharmaceutical product produced in the cell culture. This is of particular relevance if the cell culture products are administered to humans with immune compromise. One of the many main potential problems associated with the commonly used bovine serum supplement is the possibility of transmitting the causative agent of bovine spongiform encephalopathy (BSE) to animals / humans that come into contact with the products produced from cell culture.

In view of the possible risk associated with the use of animal sera in cell culture, it became evident that production processes exempt from the use of animal products are highly desirable.

To this end, specific means have been developed that do not have to be supplemented with animal sera to continuously grow cell lines and to produce viruses in continuously growing cell lines, respectively. An example of such a serum-free medium that can be used to grow cell lines is the

VP-SFM produced by Gibco BRL / Life Technologies. According to the

training of manufacturers, VP-SFM is specifically designed for the cultivation of VERO, COS-7, MDBK, Hep2, BHK-21 and other important cell lines (Price, P. and Evege, E. Focus 1997, 19: 67 -69) and for viral production in said cell lines. There is no information available regarding the cultivation of primary cells in said medium.

U.S. 5,503,582 describes the cultivation of CEF cells in medium comprising 4% calf serum followed by infection of the cells with poultry poxvirus in the serum-free medium. Spataro, A. C. et al., J. Cell. Know. 1976; 21,407-413 describe that CEF cells can be kept in serum-free medium for 24 hours once a monolayer has been formed. Thus, according to both publications,

media that are used for culturing the cells after the inoculum comprise serum. Only the serum-free medium was used for the maintenance of cells that are already adhered to the surface and that reached the stationary phase. If the inoculum was made with conventional medium, such as medium 199 or

RPMI-1640 that does not contain serum, the monolayer is not formed and the cells form non-viable aggregates in the media.

WO 98/15614 refers to a specific serum-free medium for the in vitro culture of animal cells. The cells that can be grown in the medium that is described in WO 98/15614 are those of animal origin, 30 including cells obtained from mammals, birds, insects or fish. Mammalian cells can be primary cells of human origin. There is no reference to primary bird cells.

U.S. 5,405,772 describes a serum-free medium for the proliferation

fermentation and cell development. The cells are preferably hematopoietic cells and bone marrow stromal cells. Primary bird cells are not mentioned.

WO 98/04680 describes a serum-free medium for culturing adhesion-dependent mammalian cells, such as BHK, Vero or MRC-5 cell lines. WO 98/04680 does not refer to primary cells or to bird cells.

Purpose of the Invention

It is the aim of the present invention to provide a method that allows the cultivation of primary cells, in particular primary bird cells in serum-free medium, in which the method allows (I) the cultivation of cells during the log phase and (II) the maintenance of cells in the stationary phase. In addition, if the cells are adherent cells, the medium may preferably allow (III) adherence of the adherent cells to the surface of the cell culture vessel. It is a further object of the present invention to provide a method for the production of viruses through the use of primary cells under serum-free conditions.

Detailed Description of the Invention

The present invention provides a method for growing cell

primary cells, characterized by the fact that the cells are grown in a serum-free medium that comprises a factor selected from the group consisting of growth factors and adhesion factors.

According to the present invention, primary cells that naturally grow in the form of adherent cells adhere to the surface of the cell culture vessel after the inoculum and grow in a logarithmic phase until a monolayer is formed. In accordance with the present invention, resting cells can be maintained in the medium used during cell adhesion and log growth.

The method of the present invention is not restricted to cells that form monolayers. According to an alternative embodiment, the method according to the present invention can be used for all other types of primary cells, such as cells that grow naturally in suspension culture (for example, lymphocytes or other types of blood cells) or cells that would naturally grow in the form of adherent cells, but that have been adapted to grow in suspension culture.

As shown below, the cells can also be used for serum-free virus amplification that could be useful as vaccines.

It was unexpected that primary cells that grow naturally

as adherent cells (I) can adhere efficiently to the surface of the cell culture vessel without forming unacceptable amounts of aggregates and (II) can be grown in the log phase in the absence of serum since it is generally believed that primary cells were dependent on a large number of different factors and components comprised in the serum. In addition, adherent cells were believed to form non-viable aggregates that did not adhere to the surface of the cell culture vessel when cultured in serum-free medium. Thus, it was unexpected that it would be sufficient to add to a serum-free medium a factor selected from the group consisting of growth factors and adhesion factors in order to obtain adhesion and growth of

adherent primary cells. In addition, it was still unexpected that primary cells grown in the suspension culture could grow with the media used in the method according to the present invention. Furthermore, it is surprising that primary poultry cells, such as Chicken Embryonic Broblast (CEF) cells, can be cultured to adhere to the surface of a cell culture vessel without forming unacceptable amounts of aggregates in a free medium. of serum that comprises a factor selected from the group consisting of growth factors and adhesion factors since it was known that poultry cells grew extremely poorly in serum-free medium that did not include growth factors or growth factors. adhesion, that is, it was unexpected that the poor growth properties of primary cells

birds could be significantly improved by adding a factor selected from growth factors and adherence factors to the serum-free medium.

The term primary cells as used in the present description is well known to one skilled in the art. Without being restricted to the following definition, the term primary cells refers to cells that have been newly isolated from an animal or human tissue, organ or organism, in which the cells are unable to replicate and divide continuously and indefinitely. Generally, primary cells divide into cell culture

squids less than 100 times, often less than 50 times, often less than 25 times. Thus, the primary cells did not undergo an immortalization event. Examples of primary cells are cord blood lymphocytes and human or animal fibroblasts. Preferred examples for animal fibroblasts are poultry fibroblasts, more preferably Chicken Embryonic Fibroblasts (CEF cells). A preferred example for primary human fibroblasts is fibroblasts in the human foreskin.

Methods of how primary cells can be isolated are well known to the person skilled in the art. Generally, primary cell cultures are derived directly from tissues, organs or embryos.

Tissues, organs or embryos are subjected to treatment with protease to obtain isolated cells. The cells are then cultured according to the method according to the present invention under in vitro culture conditions.

More specifically, CEF cells are obtained from protease-digested chicken embryos. According to the present invention, CEF cells grow better than adherent cells adhered to a solid cell support surface. The cells begin replication and establish a monolayer. If CEF cells (after digestion of the embryo) are cultured in vitro with a standardized culture medium without animal serum, the cells will occasionally adhere to the solid cell support surface, but will not replicate until they form a monolayer

confluent cells and will detach over time from the solid cell culture support surface. In contrast, if CEF cells are cultured according to the method of the present invention, the cells adhere to the solid support, grow in the log phase until a monolayer 5 is formed and can be maintained in the stationary phase for several days.

The term cell culture in a serum-free medium in the context of primary adherent cells refers to the inoculation of cells in the culture vessel in a serum-free medium, for cell growth in a serum-free medium in the log phase until that a single

formed and / or for maintaining cells in serum-free medium once the monolayer is formed. More preferably, the term cell culture in a serum-free medium refers to a method in which all the steps mentioned above are performed with serum-free medium, so that no animal serum product is present during the entire process. cell culture. Thus, in a more general meaning, the term cell culture in a serum-free medium refers to the fact that all of the media that lead to the formation of a monolayer are serum-free media. The means used in all the previous steps can comprise a factor selected from the growth factors and from the adhesion factors. However, it could be sufficient to add such a factor

only to the media used for cell adhesion and / or cell cultivation under logarithmic conditions.

The term cell culture in a serum-free medium in the context of cells growing in suspension culture refers to the inoculation of cells in the culture vessel in a serum-free medium, the cultivation of cells in a serum-free medium in the logarithmic phase and / or the maintenance of cells in serum-free medium as soon as the saturation density in which no further replication occurs is obtained. More preferably, the term cell culture in a serum-free medium refers to a method in which all the steps mentioned above are performed with serum-free medium, so that no animal serum product is present during the entire culture of the cells. cells. The means used in all the previous steps can preferably comprise a factor selected from the group of growth factors. However, it could be sufficient to add such a factor only to the media used for cell inoculation and / or cell growth under logarithmic conditions.

As explained in more detail below, it could still be possible to grow cells that would normally grow in the form of adhered cells, also in the form of suspension culture cells if chosen

appropriate incubation conditions (for example, by applying wave incubation). The method according to the present invention also applies to this type of incubation.

The term serum-free medium refers to any cell culture medium that does not contain sera of animal or human origin. Suitable cell culture media are known to the person skilled in the art. These media include salts, vitamins, buffers, energy sources, 15 amino acids and other substances. An example of a suitable medium for serum-free cultivation of CEF cells is medium 199 (Morgan, Morton and Parker; Proc. Soc. Exp. Biol. Med. 1950, 73, 1; which can be obtained inter alia in Life Technologies).

The media used in accordance with the method of the present invention, in particular, the media used for adherent cells such as cells

CEF squids, contain a factor selected from the group consisting of growth factors and adhesion factors. An example for an adherence factor is fibronectin.

For cells that would naturally grow in the form of adherent cells, which, however, are nevertheless grown in suspension culture (which is possible, for example, for CEF cells), it is particularly preferred to use a factor selected from the group of growth factors. Examples of useful growth factors for this type of culture are bovine, murine, chicken or human recombinant epidermal growth factor (EGF). Human recombinant EGF (rh-EGF) (Chemicon Int., Catalog number: GF001) is most preferred.

For cells that grow naturally in the culture in suspension

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pension, the medium can preferably comprise a factor selected from the group of growth factors including EGF. More preferably, the growth factors for this type of cells are specific factors for non-adherent cells. Examples of these growth factors are interleukins, GM-CSF, G-CSF and others. The person skilled in the art can easily determine through routine experiments, which type of factor is suitable for each type of cell.

If the factor added to the serum-free medium is EGF, in particular rh-EGF, it is preferably added to the medium in a concentration of 1 to 50 ng / ml, more preferably 5 to 20 ng / ml. However, the person skilled in the art will be aware of the fact that different cell types may require a slightly different concentration of EGF in the serum for optimal results.

If the adhesion factor added to the serum-free medium is fibronectin: (eg Chemicon Int .; Human plasma fibronectin; catalog number FC010), it is preferably added to the medium in a concentration of 1 to 50, more preferably of 1 to 10 pg / cm ² of surface of the cell culture vessel. However, the person skilled in the art will be aware of the fact that different cell types may require a slightly different concentration of fibronectin in the serum to re-

great results.

According to the present invention, it is sufficient to add only one factor selected from the growth factors and the factors of adhesion to the medium, particularly if the cells are adherent cells. However25, it is still possible to add two or more factors selected from the growth factors and the adherence factors to the medium. The medium can preferably comprise EGF and fibronectin, preferably in the concentration ranges defined above, particularly if the primary cells are adherent cells such as CEF cells.

The medium may further comprise one or more additives selected from microbial extract, plant extract and extract from a non-mammalian animal. The microbial extract is preferably the yeast extract or an ultrafiltered yeast extract. The vegetable extract is preferably rice extract or soy extract. The extract of a non-mammalian animal is preferably a fish extract.

According to a preferred embodiment of the present invention, Asparagine can be added to the commercially available serum-free medium to which a factor selected from growth factors and adhesion factors has been added. Most preferably, asparagine is added to the medium that is used during infection with the virus (see below). Commercial serum-free media generally comprises asparagine

in a concentration range of 0.3 to 1.0 mM. Preferred amounts of asparagine to supplement the medium are in the range of 0.5 to 1.5 mM. A 1 mM asparagine supplement is most preferred. The total concentration of asparagine in the medium is preferably less than 2 mM and more preferably in the range of 0.8 to 1.8 mM. The most preferred concentration of asparagine in the medium is 1.3 mM.

In addition, glutamine can preferably be added to the medium. Most preferably, glutamine is added to the medium that is used during infection with the virus (see below). Preferred amounts of glutamine that will be supplemented in half are in the range of 1 to

5 mM, more preferably in a range of 2 to 4 mM. The ranges indicated

also correspond to the total preferred concentrations in the medium since most of the commercially available media do not contain glutamine.

According to an additional embodiment, the invention relates to a method for amplifying a virus that comprises the following steps: in the first step, the primary cells are cultured according to the method described above, that is, the cells primaries are grown in a serum-free medium that comprises a factor selected from the group consisting of growth factors and adhesion factors, depending on the cell type. All conditions, definitions, preferred modalities and the order of preference to the most preferred modalities provided for the description of the method for the cultivation of primary cells above, size 12

They also apply to the definition of the first stage of the method for amplifying the virus according to this embodiment of the present invention. In a second step, the primary cells are infected with the virus. In the third stage, the infected cells are incubated in serum-free medium until the viral progeny is produced.

The term amplification of a virus is used to make it clear that the method according to the present invention is preferably used to increase the amount of virus because of a productive viral replication of the virus in infected cells. In other words, the ratio of virus output to virus input should preferably be greater than 1. Thus, according to the present invention these primary cells are chosen for a specific virus, in which the virus is able to replicate productively. The term reproductive replication refers to the fact that the specific virus replicates in the specific primary cell to an extent that the infectious viral progeny is produced, in which the ratio of virus output to virus input is greater than 1 .

The person skilled in the art knows, which viruses can be replicated productively in which type of primary cells. For example, primary cells may be human foreskin fibroblasts if the virus to be amplified is the human cytomegalovirus; primary cells can be CEF cells if the virus to be amplified is measles virus, mumps virus, rabies virus, Japanese encephalitis virus, yellow fever virus, influenza virus or a poxvirus such as vaccinia, in particular the modified Ankara vaccinia virus (MVA).

Methods for infecting primary cells according to the second stage of the present embodiment are known to those skilled in the art. For example, the virus can simply be added to the medium. Alternatively, the medium can be removed and the virus can be added to the fresh medium, which in turn is added to the cells. To obtain an efficient infection, the amount of virus / medium suspension must be as small as possible to have a high virus concentration.

After the virus has adhered, an additional medium can be added.

In the third stage, the cells are inoculated in serum-free medium until a viral progeny is produced.

The serum-free medium that is used in the second and third stages of the method for amplifying a virus can be the same medium that had been used previously, that is, a serum-free medium that comprises a factor selected from the growth and adherence factors, depending on the cell type. However, for economy, it is possible to use in the first or both in the second and third stages a serum-free medium that does not contain a factor selected from the

growth and adherence factors.

During all stages, the medium can be supplemented with asparagine and / or glutamine as mentioned above, where the total concentration of asparagine in the medium is preferably as defined above.

The viral progeny can be concentrated and purified according to the methods known to the person skilled in the art.

According to a preferred embodiment, the method for amplifying viruses is used for amplifying poxviruses. Thus, according to this preferred embodiment, the invention relates to a method for the amplification of a poxvirus that comprises the following steps: (I) 20 the cultivation of primary cells according to the method described above.

that is, a method in which the primary cells are grown in serum-free medium that comprises a factor selected from the group consisting of growth factors and adhesion factors, depending on the cell type, (II) the infection of the primary cells with poxvirus and (III) the cultivation of infected cells in serum-free medium until the viral progeny is produced.

It was unexpected that poxviruses could be amplified in cells cultured under serum-free conditions since the cells grow very poorly with the known serum-free medium. Thus, the additional stress associated with a poxvirus infection was expected to kill already stressed cells before significant poxvirus amplification occurred.

Ο poxvirus is preferably an orthopoxvirus. The <ρ> examples:

of orthopoxvirus are avipoxvirus and vaccinia virus.

The term avipoxvirus refers to any avipoxvirus, such as poultry poxvirus (Fowlpoxvirus), canary poxvirus, Un5 copoxvirus, Mynahpoxvirus, pigeon poxvirus, parrot poxvirus, quail poxvirus, pox virus, quail poxvirus, pox virus, quail pox virus. of peacock, penguin poxvirus, sparrow poxvirus, starling poxvirus and turkey poxvirus. The preferred avipoxviruses are canary poxvirus and poultry poxvirus.

An example of a canary poxvirus (Fowlpoxvirus) is the strain

Rentschler. A plaque purified canary poxvirus strain called ALVAC (U.S. 5,766,598) was deposited under the terms of the Treaty of

Budapest at American Type Culture Collection (ATCC), accession number

VR-2547. Another strain of canary poxvirus is the commercial canary poxvirus vaccine strain called LF2 CEP 524 24 10 75, available from Institute Merieux, Inc.

Examples of a poultry poxvirus are strains

FP-1, FP-5 and TROVAC (U.S. 5,766,598). FP-1 is a modified Duvette strain to be used as a vaccine in day old chickens. The strain is a vaccine strain of commercial poultry poxvirus 20 called O DCEP 25 / CEP67 / 2309 October 1980 and is available at

Institute Merieux, Inc. FP-5 is a vaccine strain of chicken poultry commercial poxvirus of embryonic origin available from American

Scientific Laboratories (Division of Schering Corp.) Madison, Wisconsin, United States Veterinary License No. 165, ^N's 30321 series.

Examples of vaccinia virus strains are the Temple of

Heaven, Copenhagen, Paris, Budapest, Dairen, Gam, MRIVP, Per,

Tashkent, TBK, Tom, Bem, Patwadangar, BIEM, B-15, Lister, EM-63, New York City Board of Health, Elstree, Ikeda and WR. The invention is preferably carried out with the modified vaccinia Ankara virus (MVA) (Sutter, G. et al. [1994], Vaccine 12: 1032-40). A typical MVA strain is MVA 575 which was deposited with the European Collection of Animal Cell Cultures under deposit number ECACC V00120707. The most preferred is MVA-BN or a derivative

·· · • · • · ·· hereby, which was described in PCT patent application PCT / EP01 / 13628 filed with the European Patent Office on November 22, 2001, entitled, Modified Vaccinia Ankara Virus Variant. MVA-BN was deposited with the European Collection of Animal Cell Cultures under deposit number 5 ECACC V00083008. The characteristics of the MVA-BN, the description of the biological assays that make it possible to assess whether an MVA is an MVA-BN or a derivative of it and the methods that allow obtaining the MVA-BN or one described therein, are disclosed in the application for PCT patent referenced above which is incorporated herein by reference.

The virus that will be amplified according to the method of the present invention can be a wild type virus, an attenuated virus or a recombinant virus.

The term recombinant virus refers to any virus that has a heterologous gene inserted into its viral genome that is not naturally part of the viral genome. A heterologous gene can be a therapeutic gene, a gene that encodes a peptide that comprises at least one epitope to induce an immune response, an antisense expression cassette or a ribozyme gene. The constructive methods of a recombinant virus are known to one skilled in the art. The most preferred poxvirus vector 20 is MVA, in particular MVA 575 and MVA-BN (see above).

An attenuated virus is a virus that originates from a pathogenic virus, but which after infection of the host organism leads to less mortality and / or morbidity compared to that of the original unattenuated virus. Examples of attenuated poxviruses are known to the person skilled in the art. An example for an attenuated Vaccinia virus is the MVA strain, in particular the strain that was deposited at ECACC under the deposit number V00083008 (see above).

As previously mentioned, for poxviruses the primary cells can preferably be primary cells of birds such as CEF cells or primary duck embryonic fibroblasts. Again, the skilled person is aware of which primary cells are suitable for the amplification of which poxvirus. CEF cells are particularly preferred for the amplification of MVA. For the amplification of MVA in CEF cells, it is a preferred modality to select one or two of the selected factors of EGF and fibronectin.

If the method according to the present invention is used for the amplification of MVA in CEF cells, it is preferred that the initial pH of the medium is in a range of approximately 7.0 to approximately 8.5. An initial pH of 7.0 is particularly preferred.

The invention also relates to viruses, in particular to poxviruses obtained by the method described above. According to a preferred embodiment, the poxvirus is a vaccinia virus, more preferably an MVA strain such as MVA-BN.

The invention also relates to a composition that comprises a virus, in particular a poxvirus produced by the method according to the present invention. As mentioned earlier, the poxvirus is preferably a vaccinia virus, more preferably an MVA strain such as MVA-BN. Because of the method for amplifying the virus, the composition is free of any infectious products and / or agents contained in animal sera. In contrast, compositions comprising viruses produced according to conventional methods comprise

residual posts derived from animal serum. This is especially the case for compositions comprising poxviruses such as vaccinia virus strains.

The invention further relates to a virus, in particular to viruses that are defined above as a medicine or vaccine. If the virus is a wild type virus or an attenuated virus, the virus can be used for vaccination against the virus as such. For this purpose, attenuated viruses are particularly preferred. If the virus is a recombinant virus that expresses proteins expressed from genes heterologous to the viral genome, it is possible to vaccinate against the virus as such and / or against the expressed heterologous protein. If the recombinant virus expresses a therapeutic gene, such as an antisense RNA or a ribozyme, the virus can be used as a medicine.

The invention also relates to the use of a virus as defined

previously or to a composition as previously defined for the production of a vaccine. The invention also relates to a method for the treatment or 5 for vaccination of an animal including a human being in need thereof, which comprises administering a virus as defined above or a composition as defined above to the animal or human body. Examples If not indicated otherwise, in the following examples, the serum-free medium is medium 199 (Life Technologies). The added EGF is generally the recombinant human EGF obtained from Chemicon. Fibronectin (FN) was obtained from Chemicon. Example 1: Preparation of Chicken Embryo Fibroblast (CEF) cells 15 Fertilized eggs free of specific pathogens (SPF) were stored for no more than 12 days at 4 ° C. The eggs were placed in an incubator and incubated for 10-12 days at

37.8 ° C + 8 ° C. A petri dish for a maximum of 11 eggs was prepared with 10-20 ml of PBS. The eggs were placed in a cardboard box dedicated to the eggs and treated extensively with Bacilol to sterilize the

outside of the eggshell. After drying, a hole was made in the eggs and the shell was removed carefully. The chorioallantoic membrane was removed. The embryos were lifted by the feet and then their heads were cut off. The embryos were then transferred to prepared petri dishes. After removing the feet, the logs were washed again with PBS. A maximum of 11 logs were placed in a 20 mL plastic syringe and squeezed into an Erlenmeyer flask. 5 ml of preheated (37 ° C) Trypsin / EDTA solution per stem was added and the solution was stirred for 15 minutes with serum-free medium at room temperature using a magnetic stirrer. The cells subjected to trypsin treatment were poured through a mesh layer in a beaker. All cells were transferred to a centrifuge tube

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225 mL and centrifuged at 20 ° C, 470xg for 7 minutes in a bench top centrifuge. After discarding the supernatant, the pellet was resuspended in 1 mL of growth medium free of pre-heated fresh serum (37 ° C) comprising 10 ng / mL of EGF per stem through vigorous pipetting up and down. Preheated (37 ° C) fresh serum-free growth medium comprising 10 ng / ml EGF was added to a total volume of 150 ml. The centrifugation step was repeated. The supernatant was removed and the pellet was resuspended as described

previously. Preheated (37 ° C) fresh serum-free growth medium comprising 10 ng / ml EGF was added to a total volume of 100 ml. The cells were counted as described in the following section. The required amounts of cells were inoculated into spinner bottles with serum-free growth medium comprising 10 ng / ml EGF and incubated at 37 ° C. The cells were ready for viral infection on the 15th day after inoculum.

Example 2: Cell Density Count

A sample of the cell suspension (see the CEF preparation section) was taken and mixed with a volume of Trypan blue, resulting in a final cell count of 20 to 100 cells per 16 small blocks of a hemocytometer provided by Fuchs- Rosenthal under the

Hemocytometer Fast Read 102 (1: 2 dilution - T.10). The sample was taken immediately after resuspending the cells in order to prevent cell re-aggregation / sedimentation. After a few minutes of the incubation time with Trypan blue in order to acquire the stain properly in the dead cells, 10 pL of the cell suspension was added to the hematocytometer. Only white living cells were counted under a light microscope using a 10x objective. In total, 3 large representative squares consisting of 3x16 small were counted. Of each large square, only two edges in the L-shape were included in the count. The counted cells were averaged and the final cell concentration was calculated using the following formula: Average number of cells x dilution x 10 ⁴ = cells / mL. Finally, the

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cell suspension was diluted to the desired working concentration. Example 3: Effect of adding a selected factor of growth factors and fibronectin to a serum-free culture medium on the formation of a CEF monolayer

In preliminary experiments, the inventors showed that CEF cells do not adhere to the surface of cell culture vessels if medium 199 that does not comprise FCS is used. In addition, no monolayer is formed. The formation of normal monolayers is observed if medium 199 containing 7% FCS is used. It was analyzed whether

healthy and growth of CEF cells in serum-free medium 199 could be achieved if additives were added to the medium.

The tested additives comprise the recombinant Epidermal Growth Factor (r-hEGF) and Fibronectin (FN).

For the experiments, CEF cells were grown in a medium

199 with different additives alone or in combination. The cells grown in medium 199 without any additives served as the negative control. Cells grown in medium 199 comprising 7% FCS served as the positive control. All experiments were performed in 6-cell cell culture plates with 3 mL of medium. Additives 20 were treated according to the supplier's data sheets before

be used for cell culture. Fibronectin was allowed to be absorbed by the surface of the cell culture plates for 25 minutes before use. Fibronectin was used at a concentration of 3 pg / cm ² and EGF was used at a concentration of 10 ng / ml. Before adding any cells, the cell culture plates were placed in contact with the medium containing fibronectin for 25 minutes.

Each culture medium plus the additives to be tested was grown in duplicates. The 6-well cell culture plates were incubated for 4 days at 37 ° C. From 1 ^s to 4 ^S days, cell adhesion and growth were assessed using a microscope.

For positive control, normal CEF cell adhesion and growth was observed. For Meio 199 without additives, almost ne20 dl

no adhesion of CEF cells could be observed.

A crucial increase in the formation of a monolayer was observed through the use of EGF added to Medium 199 compared to Medium 199 without additives. It was discovered that the cells adhered and formed the typical fibroblast morphology. In addition, continuous growth could be observed over the entire 4-day period.

An increase in cell adhesion was also achieved by adding fibronectin to the culture medium. The addition of both EGF and Fibronectin resulted in a slight increase compared to the addition of only EGF and only Fibronectin.

In summary, it was concluded that the formation of the CEF cell monolayer in Medium 199 serum-free can be sustained by the use of the additives EGF and Fibronectin.

In addition, in parallel sets of experiments, 1 x 10 ⁷ cells of CEF were inoculated in the medium comprising 10% FCS, in the medium not comprising FCS and in the medium not comprising FCS, but comprising EGF. The number of cells was counted 2 days after the inoculum. The number of cells was equivalent to 42%, 6% and 44%, respectively, of the number of cells used for the inoculum. Thus, the results for cells inoculated in serum-free medium comprising EGS were as good as the results obtained with medium comprising FCS and significantly better than those with medium containing no serum or EGF.

In addition, the medium comprising EGF has been compared with several standard serum-free media, such as DMEM, Opti-Mem or 293-SFM. For this purpose, 1 x 10 ⁷ CEF cells were inoculated into the various serum-free media and cultured for 4 days. The number of cells cultured in medium comprising EGF was 24.5 and 12 times greater than the number of cells cultured in DMEM, Opti.Mem and 293-SFM serum-free, respectively.

Example 4: Infection of CEF cells with MVA

CEF cells were infected four days after inoculation in spinner bottles. At such a point in time, the cells had grown to

- a suitable monolayer. The cells were infected with an MOI of 1 or 0.1 MVA. For infection, the culture medium was removed from the flasks. The desired amount of virus per spinner bottle was diluted in 20 ml of the appropriate serum-free infection medium. At this stage, the medium 5 serum-free may or may not comprise a factor selected from growth factors and fibronectin. The cells were incubated with the virus for 1 hour at 37 ° C at 0.3-0.5 rpm in a rotary bottle incubator. After 1 hour, the spinner bottles were filled with the appropriate serum-free growth medium in a final volume of 200 mL per bottle ¹⁰ rotating. At this stage, the serum-free medium may or may not comprise a factor selected from growth factors and fibronectin. Viral replication was stopped after 48 or 72 hours by freezing the revolving bottles to -20 ° C. Example 5: Preparation of Viral Extracts from infected CEF Cells . * 15 and Titration of the MVA The frozen spin bottles were thawed at room temperature. During the defrosting process, the cells detached from the surface of the spinner bottles and could be mechanically removed by shaking the flasks. Viral / cell suspension 20 and was collected and aliquoted in smaller volumes. For virus release from infected cells, virus / cell suspensions were frozen / thawed 3 times. Frozen / thawed virus samples were used for titration. Titrations were performed in the 1- passage of CEF cells 25 in 96-well plates, using 10-fold dilutions of the viral suspension and 8 replicates per dilution. After infection, the infected cells were visualized with a vaccinia antivirus antibody and an appropriate staining solution. In detail, on day zero of the assay, the primary CEF cells 30 (see the section Preparation of Chicken Embryonic Fibroblasts (CEF) cells) were subjected to trypsin treatment and counted as described in the cell density counting section. The cells were diluted

up to 1x10 ⁵ cells / mL in RPMI medium with 7% FCS. After this dilution, 100 gL were inoculated into each well of the 96 well plates using a multichannel pipette. The cells were incubated overnight

37 ° C and 5% CO2. The virus samples to be titrated (see the section preparing viral extracts from infected CEF cells) were serially diluted in 10-fold steps of 10 ' ¹ - 10 ¹² using RPMI without serum. This serial dilution is performed by adding 900 μΙ_ of

RPMI in all wells of a 96-well deep plate. 100 μΙ_ of the viral sample was added to all wells in the first row and 10 were mixed. After that, 100 μΙ_ of each sample was transferred to the next row of wells using a multichannel pipette. The 96-well deep plates were kept on ice while dilutions were performed. The plates were incubated for 5 days at 37 ° C and 5%

CO2 to allow the infection to continue. After 5 days, the cells were stained immunohistochemically with an antibody specific for the vaccinia virus. For staining, the culture medium was removed by turning the 96-well plate upside down on a recipient. The cells were fixed with 100 µl / well of methanol / acetone mixture (1: 1) for 10 minutes at room temperature. The fixing solution was removed and the plates were air-dried. After drying, the cells were washed once with PBS and incubated for 1 hour at room temperature with the vaccinia antivirus antibody (rabbit polyclonal IgG fraction of the Vaccinia antivirus antibody (Quartett, Berlin, Germany No. 9503-2057) diluted in

1: 1000 in PBS with 3% FCS. After removing the antibody, the cells were washed twice with PBS and incubated for 1 hour at room temperature with anti-rabbit antibody coupled with HRP (coupling with Peroxidase from Strong Root) (anti-rabbit IgG antibody, polyclonal goat coupled with HRP (Promega , Mannheim, Germany n ^and W4011) diluted 1: 1000 in PBS with 3% FCS. Again, the cells were washed with PBS and stained with o-Dianisidine or TMB. To use the staining method with o-Dianisidine , cells were incubated with 100 μL / well of staining solution consisting of 5 mg o-Dianisidine

- and 180 μί of 30% H2O2 per 60 mL of 50 mM phosphate-citrate buffer. The cells were incubated at room temperature until they were stained brown. The infected cells were clearly visible after 1-3 hours. Using the TMB staining method, cells were incubated with 5 30 pL / 1.2 mM well of TMB (Seramun Diagnostica GmbH). After 15 minutes of the incubation time, the TMB solution was removed and the cells were washed once with PBS. The infected cells appeared as dark blue. The plates were classified in relation to the infected cells. The viral titer was calculated using the Spearman and Kaerber formula. 10 • For the TCID50 calculation, each well showing brown or blue cells was marked as positive. Due to the fact that the assay parameters are kept constant, the following simplified formula was used: Viral title [TCIDso / mL] = 1 ₀ ^{[a + 1} - ^{5 + xa / 8 + xb / 8 + xc / 8]} a = dilution factor of the last column, in which all eight cavities 15 are positive x _a = number of positive wells in column a + 1 Xb = number of positive wells in column a + 2 x _c = number of positive wells in column a + 3 Example 6: Optimal inoculum density for CEF cells in middle 20 serum-free and optimal amount of MVA for infection of CEF cells and An optimal cell density for the inoculum of 7.5x10 ⁷ cells / 850 cm ² (surface of a spinner flask) was determined for the growth of serum-free CEF. The cells were able to build a good monolayer without forming large lumps on the fourth day after the inoculum 25 and could be infected at this point in time. The experiments were carried out to determine the best level of viral inoculation and the period of infection for maximum production of MVA from CEF cells cultured in a serum-free process. CEF cells were seeded at a density of 7,5x10 ⁷ cells / 850 cm ² 30 in medium according to the present invention. At 4 ^S day after the inoculum, the cells were infected with different amounts of MVA in the range of 0.05 to 1.0 TCIDso / MVA cell. The best results were obtained

• ··· • · • · with 0.1 TCID ₅₀ / MVA cell.

Example 7: Optimal pH of serum-free medium for cultivation and infection with

MVA

Infections with MVA and other poxviruses are sensitive to a pH below 7.0. Poxviruses are not stable at acidic pH and it is recommended that purified poxviruses be stored in a buffered solution with a pH greater than 7.0 to ensure viral stability and integrity after storage in the form of a liquid viral preparation. The experiments were carried out to determine the effect on the yield of

virus when an infection occurs at a different starting pH. Spinner flasks were inoculated with CEF cells in the usual manner in serum-free medium comprising 10 ng / ml EGF plus 4 mM L-Glutamine and cultured for 4 days. The cells were infected with 0.1 MVA

TCID ₅₀ / cell in serum-free medium comprising 10 ng / mL EGF 15 plus L-Glutamine and 1 mM Asparagine at different pHs ranging from 6.5 to 9.0. 72 hours after infection, the pH of the medium was measured and the viral yields were determined by titrating the cell extracts in the usual manner. The results are shown in the table below, which shows the effect of the initial pH of the medium at the beginning of the infection on the viral yield.

Initial pH.

serum-free medium comprising 10 ng / ml EGF Initial pH pH 72 h p.i. Title [TCID5o / mL] 6.5 7.05 0.56 X 10 ⁷ 7.0 7.34 10.0 X10 ⁷ 7.5 7.3 5.60X10 ⁷ 8.0 7.68 8.60 X10 ⁷ 8.5 7.75 7.80 X 10 ⁷ 9.0 8.03 0.65 X 10 ⁷

For infections carried out in serum-free medium comprising 10 ng / mL of EGF supplemented with L-Glutamine and Asparagine, viral production was relatively constant with the initial pH of 7.0 to 8.5, but viral productions were low in Initial pHs of 6.5 and 9.0. O

best yield was obtained at the initial pH of 7.0. Commercially available standard serum-free media generally has a pH of 7.4. Therefore, adjusting the pH of the serum-free medium to 7.0 can help increase viral yield.

Effect 8: Effect of Asparagine added to serum-free medium

Preliminary experiments revealed that the amount of asparagine can be limiting during the cultivation of CEF cells and the infection of CEF cells with MVA. To overcome the elimination of Asparagine in serum-free media during the culture and infection process, extra Asparagine was added to the medium as a supplement prior to infection with CEF cells. To determine the optimal amount of Asparagine to supplement the medium, the spinner bottles were inoculated with CEF cells (7.5x10 ⁷ cells / 850 cm ² ) in serum-free medium comprising 10 ng / mL EGF plus 4 mM L-Glutamine . Four days after inoculation, the cells were infected with MVA at 0.1 TCID ₅₀ / cell in serum-free medium comprising 10 ng / mL EGF plus 4 mM L-Glutamine supplemented with different concentrations of Asparagine (0.5 , 1.0 and 1.5 mM). Viral replication was stopped 72 hours after infection and viral titers were determined. The results are shown in the table below which shows MVA production from CEF cells supplemented with different levels of Asparagine for the infection stage. The titles represent the averages of 3 revolving bottles supplemented with Asparagine.

Asparagine Supplement Viral titers after 72 hours of infection [TCIDso / mL] 0.0 mM 1.8X10® 0.5 mM 1.3X10® 1.0 mM 6.8X10® 1.5 mM 1.0X10®

The results demonstrate that supplementation of serum-free medium comprising 10 ng / mL of medium with EGF and Asparagine could increase viral production and that supplementation by 1 mM for the infection process was optimal.

Claims (3)

1. Method for amplifying a poxvirus characterized by the fact that it comprises the following steps: (a) to cultivate embryonic chicken fibroblasts (CEF) in a serum-free medium; (b) infect chicken embryonic fibroblasts (CEF) with the poxvirus; and (c) cultivating the infected cells in serum-free medium until the poxvirus progeny is produced, in which chicken embryonic fibroblasts (CEF) are cells that allow productive replication of the poxvirus, and in which the serum-free medium from steps (a) to (c) comprise epidermal growth factor (EGF). 2. Method, in wake up with The claim 1, characterized by fact that EGF is Recombinant EGF- human. 3. Method, in wake up with The claim 2, characterized by the fact that the concentration of EGF is in a range of 5 to 20 ng / ml of medium. Method according to any one of claims 1 to 3, characterized in that said serum-free medium of steps (a) to (c) further comprises an adhesion factor, preferably fibronectin. 5. Method according to claim 4, characterized by the fact that the concentration of fibronectin is in the range of 1 to 10 pg / cm ² of surface of the cell culture vessel. 6. Method, according to claims 1 to 5, characterized by the fact that the medium comprises two or more factors selected from growth factors and Petition 870170049703, of 7/17/2017, p. 14/16 2/3 adhesion. Method according to claim 6, characterized in that the medium comprises EGF and fibronectin in concentration ranges as defined in claims 4 and 6. Method according to any one of claims 1 to 7, characterized in that the medium further comprises one or more additives selected from microbial extract, plant extract and extract from a non-mammalian animal. 9. Method according to claim 8, characterized by the fact that the microbial extract is a yeast extract or an ultrafiltered yeast extract. 10. Method, in wake up with the claim 8, characterized by the fact that the extract vegetable is an extract of rice or extract in Soy. 11. Method, in wake up with the claim 8, characterized by fact that the extract of an animal no mammal is a fish extract. 12. Method, in wake up with Any of them of claims 1 to 11, characterized by the fact that the poxvirus is an orthopoxvirus. 13. Method according to claim 12, characterized by the fact that the orthopoxvirus is a Vaccinia virus. 14. Method according to claim 13, characterized by the fact that the Vaccinia virus is Modified Vaccinia Ankara virus. Method according to any one of claims 12 to 14, characterized in that the poxvirus is an attenuated virus or a recombinant virus. Petition 870170049703, of 7/17/2017, p. 15/16 3/3 16. Method according to any one of claims 1 to 15, characterized in that subsequent to the stage of cultivation of infected cells in serum-free medium, one or more purification steps are carried out until the production of poxvirus progeny is produced .